Adenovirus with modified binding moiety specific for the target cells

ABSTRACT

An adenovirus or adenovirus-like particle which has a modified binding specificity conferred by a binding moiety. The binding moiety is heterologous to the adenovirus and is incorporated as a fusion protein with the fiber protein. This allows the adenovirus or adenovirus-like particle to bind to a target cell which is not the natural host cell of the virus. The penton fiber is modified by the insertion or, deletion, or substitution of amino acid residues, that disrupt the host-cell binding function so that the adenovirus or adenovirous like particle does not bind the natural host cell.

This Application is the national stage filing under 35 U.S.C. 371 ofPCT/GB93/02267, which was filed on Nov. 4, 1993, published as WO94/10323May 11, 1994.

The present invention relates to delivery vehicles for genes to targetcells, especially in the fields of gene therapy and cancer treatment.

The delivery of genes to target cells, especially those within themammalian body, has many uses, for example in the fields of genetherapy, cancer treatment and in areas of genetic manipulation still tobe discovered. The gene to be delivered may encode a molecule, such as aprotein or RNA, which is cytotoxic to the target cell, or it may encodea functional copy of a gene that is defective in the target cell. Inthis latter case the product of the aforementioned functional copy ofthe gene will replace that of the defective copy, and the target cellwill be able to perform its proper function.

The use of viruses, or virus-like particles, to deliver genes for genetherapy and cancer treatment has been disclosed.

However, in most cases the targeting of the virus or virus-likeparticles containing the desired gene to the cell has relied on thenatural host-virus specificity or on local application of the virus tothe cells to be targeted, for example direct application of viruses tolung cells by inhalation.

The human adenovirus 5 (Ad5) genome consists of a double-stranded linearDNA molecule of 36 kilo-basepair. The virus replication cycle has twophases: an early phase, during which four transcriptional units E1, E2,E3, and E4 are expressed, and a late phase occurring after the onset ofviral DNA synthesis when late transcripts are expressed from the majorlate promoter (MLP). These late messages encode most of the viralstructural proteins. E1, E2, and E4 gene products of human adenoviruses(Ads) are involved in transcriptional activation, cell transformation,and viral DNA replication as well as other viral functions, and areessential for viral growth. In contrast, E3 gene products are notrequired for viral replication in cultured cells or for acute lunginfection of cotton rats, but appear to be involved in evading immunesurveillance in vivo.

By "virus-like particle" we mean a nucleoprotein particle containing acore of nucleic acid surrounded by protein which (i) is not infectiveand (ii) can only be propagated in a suitable cell system followingtransformation by its nucleic acid. Thus a virus-like particle ofmammalian origin may be propagated in Saccharomyces cerevisiae or ininsect cells via a baculovirus expression system.

The modification of coat proteins of filamentous bacteriophages(bacterial viruses), such as M13 and fd, so as to generate novel bindingproperties, has been disclosed in Cwirla et al (1990) Proc. Natl. Acad.Sci. USA 87, 6378-6382 and Scott & Smith (1990) Science 249, 386-390.

It has previously been suggested that retroelement particles, includingretroviral vectors, may be modified to target specific cells, forexample see Kingsman et al (1991) Tibtech 9, 303-309.

Russell et al (1993) Nucl. Acids Res. 21, 1081-1085, published after thepriority date for this application but before the filing date disclosesretroviral vectors displaying functional antibody fragments and suggeststhat, in principle, the display of antibody fragments on the surface ofrecombinant retroviral particles could be used to target virus to cellsfor gene delivery. However, it is not known whether a retrovirus can beassembled in which all the subunits of the viral envelope protein arefused to antibody, and if so whether the virus would infect cells.NIP-derivatised human cells were tested as a method for targeted genedelivery, but became permissive for both modified (displaying ananti-NIP antibody) and unmodified ecotropic viral particles. NIP is4hydroxy-3-iodo-5-nitrophenylacetic acid.

Michael et al (1993) J. Biol. Chem. 268, 6866-6869, published after thepriority date of this application but before the filing date, describesmolecular conjugates between adenovirus and a vector system comprisingtwo linked domains, a DNA binding domain and a ligand domain. In thisconfiguration, however, it is stated that the viral moiety functions inthe capacity of both an alternate ligand domain of the conjugate and,since an additional ligand has been introduced into the conjugatedesign, the potential for cell-specific targeting is undermined.

Curiel et al (1992) Human Gene Therapy 3, 147-154 describes adenoviruseswherein a foreign epitope was introduced into the hexon protein andpolylysine-antibody complexed DNA was attached to adenovirus by virtueof the antibody binding the foreign epitope on the hexon. Foreign DNA istransferred bound to the exterior of the virion.

The above-mentioned viruses and virus-like particles may be able totarget cells using the binding moiety displayed on their surface butthey can also still target their natural host cells.

We have now devised new viruses and virus-like particles at least someof which can bind the target cell with high specificity and may delivergenetic material to the target cell; at least some of the viruses andvirus-like particles may bind and deliver genetic material to the targetcell without substantially binding to the natural host cell of thevirus.

One aspect of the present invention provides a virus, or virus-likeparticle, derived from a virus or virus-like particle having a receptorfor a host cell comprising a modified binding specificity conferred by abinding moiety allowing the virus or virus-like particle to bind to atarget cell characterised in that the said host cell receptor ismodified or absent so that the virus or virus-like particle issubstantially incapable of binding the said host cell.

By "a virus or virus-like particle substantially incapable of bindingits host cell" is included a modified virus has no more than 1% of thebinding affinity of the unmodified virus for the host cell.

In general, the binding specificity of a natural virus or virus-likeparticle is conferred by the specific interaction between areceptor-like molecule expressed on the surface of the virus orvirus-like particle and a cognate receptor-like molecule expressed onthe surface of its host cell. The invention provides a beneficialmodification of the binding specificity, so that the virus or virus-likeparticle can bind to a different specific target cell.

The introduction of the modified binding moiety may be such as toachieve the said removal of the native binding specificity.

A second aspect of the invention comprises an adenovirus or influenzavirus or vaccinia virus, or a replication-defective derivative of any ofthese, characterised in that the virus has a modified bindingspecificity conferred by a binding moiety allowing the virus to bind toa target cell.

By "binding moiety" we mean a molecule that is exposed on the surface ofthe virus or virus-like particle which is able to bind to a molecule onthe target cell. The "binding moiety" may be a molecule on the virus orvirus-like particle modified in such a way that its binding specificityis changed, or it may be a molecule added to, and exposed on the surfaceof, the virus or virus-like particle to provide a new bindingspecificity.

It is preferred if the binding moiety is external to the receptor forits host cell of the naive, unmodified virus.

It is further preferred if the binding moiety is joined or fused to thevirus or virus-like particles directly or indirectly by a spacer group.

By "host cell" we mean the cell that an unmodified, naive virus can bindto using its receptor-like molecule and the cognate receptor-likemolecule on the cell. By "target cell" we mean the cell that themodified virus can bind to using its binding moiety. In somecircumstances in the context of the second aspect of the invention, suchas when the binding moiety recognises an entity on the host cell whichis not the cognate receptor-like molecule, then the host cell may be thetarget cell.

The virus or virus-like particle may be a bacteriophage and the targetcell a bacterium in which case the invention may find uses in thetreatment of bacterial infections.

In a preferred embodiment of the invention the target cell iseukaryotic. The eukaryotic cell may be a yeast cell and the virus orvirus-like particle may be useful in the medical field in treating yeastinfections such as athlete's foot or Candida infection but it ispreferred that the eukaryotic cell is mammalian, and it is expected thatthe invention will find uses in the areas of gene therapy and cancertreatment.

In preferred embodiments of the first aspect of the invention the virusor virus-like particle is adenovirus or influenza virus or a pox-virussuch as vaccinia.

It is also preferred that the virus or virus-like particle is"replication-defective". By "replication defective" we mean a viruswhose genetic material has been manipulated so that it cannot divide orproliferate in the cell it infects.

The binding moiety of the virus or virus-like particle of the inventionprovides the target cell binding specificity. Any cell-binding proteinor peptide or carbohydrate or lipid may be useful for targeting thevirus or virus-like particle to the cell. For example, short linearstretches of amino acids, such as those constituting a peptide hormone,are useful, as are domains of polypeptides that can fold independentlyinto a structure that can bind to the target cell.

In one preferred embodiment the binding moiety has the property of anyone of a monoclonal antibody, ScFv (single chain Fv fragment), a dAb(single domain antibody) or a minimal recognition unit of an antibody.

The binding site on the target cell may be a target cell-specificantigen. Such antigens are listed in Table 1. Other binding moieties,targets on cells, and diseases which could usefully be treated usingreagents delivered by the modified viruses or virus-like particles aregiven in Table 2.

                  TABLE 1    ______________________________________    Antigen       Antibody      Existing Uses    ______________________________________    1. Tumour Associated Antigens    Carcino-embryonic                 {C46 (Amersham)                               Imaging & Therapy    Antigen      {85A12 (Unipath)                               of colon/rectum                               tumours.    Placental Alkaline                 H17E2 (ICRF,  Imaging & Therapy    Phosphatase  Travers & Bodmer)                               of testicular and                               ovarian cancers.    Pan Carcinoma                 NR-LU-10 (NeoRx                               Imaging & Therapy                 Corporation)  of various                               carcinomas incl.                               small cell lung                               cancer.    Polymorphic  HMFG1 (Taylor Imaging & Therapy    Epithelial Mucin                 Papadimitriou, ICRF)                               of ovarian cancer,    (Human milk fat            pleural effusions.    globule)    β-human Chorionic                 W14           Targeting of enzyme    Gonadotropin               (CPG2) to human                               xenograft                               choriocarcinoma in                               nude mice. (Searle                               et al (1981) Br. J.                               Cancer 44, 137-144)    A Carbohydrate on                 L6(IgG2a).sup.1                               Targeting of alkaline    Human Carcinomas           phosphatase (Senter                               et al (1988) P.N.A.S.                               85, 4842-4846    CD20 Antigen on B                 1F5 (IgG2a).sup.2                               Targeting of alkaline    Lymphoma (normal           phosphatase. (Senter    and neoplastic)            et al (1988) P.N.A.S.                               85, 4842-4846    Other antigens include alphafoetoprotein, Ca-125 and prostate specific    antigen.    2. Immune Cell Antigens    Pan T Lymphocyte                 OKT-3 (Ortho) As anti-rejection    Surface Antigen            therapy for kidney    (CD3)                      transplants.    B-lymphocyte RFB4 (Janossy,                               Immunotoxin therapy    Surface Antigen                 Royal Free Hospital)                               of B cell lymphoma.    (CD22)    Pan T lymphocyte                 H65 (Bodmer,  Immunotoxin    Surface Antigen                 Knowles ICRF, treatment of Acute    (CD5)        Licensed to Xoma                               Graft versus Host                 Corp., USA)   disease, Rheumatoid                               Arthritis.    3. Infectious Agent-Related Antigens    Mumps virus-related                 Anti-mumps    Antibody conjugated                 polyclonal antibody                               to Diphtheria toxin                               for treatment of                               mumps.    Hepatitis B Surface                 Anti HBs Ag   Immunotoxin against    Antigen                    Hepatoma.    ______________________________________     .sup.1 Hellstrom et al (1986) Cancer Res. 46, 3917-3923     .sup.2 Clarke et al (1985) P.N.A.S. 82, 1766-1770

                  TABLE 2    ______________________________________    Binding moieties for tumour-specific targets and tumour    associated antigens    Target         Binding moiety                                Disease    ______________________________________    Truncated EGFR anti-EGFR mAb                                Gliomas    Idiotypes      anti-id mAbs B-cell lymphomas    EGFR (c-erbB1) EGF, TGFα                                Breast cancer                   anti-EGFR mAb    c-erbB2        mAbs         Breast cancer    IL-2 receptor  IL-2         Lymphomas                   anti-Tac mAb and leukaemias    IL-4 receptor  IL-4         Lymphomas                                and leukaemias    IL-6 receptor  IL-6         Lymphomas                                and leukaemias    MSH (melanocyte-                   α-MSH  Melanomas    stimulating hormone)    receptor    Transferrin receptor                   Transferrin  Gliomas    TR)            anti-TR mAb    gp95/gp97      mAbs         Melanomas    p-glycoprotein cells                   mAbs         drug-resistant    cluster-1 antigen (N-                   mAbs         Small cell lung    CAM)                        carcinomas    cluster-w4     mAbs         Small cell lung                                carcinomas    cluster-5A     mAbs         Small cell lung                                carcinomas    cluster-6 (LeY)                   mAbs         Small cell lung                                carcinomas    PLAP (placental                   mAbs         Some seminomas    alkaline phosphatase)       Some ovarian;                                some non-small cell                                lung cancer    CA-125         mAbs         Lung, ovarian    ESA (epithelial                   mAbs         carcinoma    specific antigen)    CD 19, 22, 37  mAbs         B-cell lymphoma    250 kDa        mAbs         Melanoma    proteoglycan    p55            mAbs         Breast cancer    TCR-IgH fusion mAbs         Childhood T-cell                                leukaemia    Blood gp A antigen                   mAbs         Gastric and colon    (in B or O                  tumours    individuals)    ______________________________________

The binding moiety may be a monoclonal antibody. Monoclonal antibodieswhich will bind to many of these antigens are already known but in anycase, with today's techniques in relation to monoclonal antibodytechnology, antibodies can be prepared to most antigens. The bindingmoiety may be a part of an antibody (for example a Fab fragment) or asynthetic antibody fragment (for example, ScFv). Suitable monoclonalantibodies to selected antigens may be prepared by known techniques, forexample those disclosed in "Monoclonal Antibodies: A manual oftechniques", H. Zola (CRC Press, 1988) and in "Monoclonal HybridomaAntibodies: Techniques and Applications", J. G. R. Hurrell (CRC Press,1982).

Suitably prepared non-human antibodies can be "humanized" in known ways,for example by inserting the CDR regions of mouse antibodies into theframework of human antibodies.

The variable heavy (V_(H)) and variable light (V_(L)) domains of theantibody are involved in antigen recognition, a fact first recognised byearly protease digestion experiments. Further confirmation was found by"humanization" of rodent antibodies. Variable domains of rodent originmay be fused to constant domains of human origin such that the resultantantibody retains the antigenic specificity of the rodent parentalantibody (Morrison et al (1984) Proc. Natl. Acad. Sci. USA 81,6851-6855).

That antigenic specificity is conferred by variable domains and isindependent of the constant domains is known from experiments involvingthe bacterial expression of antibody fragments, all containing one ormore variable domains. These molecules include Fab-like molecules(Better et al (1988) Science 240, 1041); Fv molecules (Skerra et al(1988) Science 240, 1038); ScFv molecules where the V_(H) and V_(L)partner domains are linked via a flexible oligopeptide (Bird et al(1988) Science 242, 423; Huston et al (1988) Proc. Natl. Acad. Sci. USA85, 5879) and dAbs comprising isolated V domains (Ward et al (1989)Nature 341, 544). A general review of the techniques involved in thesynthesis of antibody fragments which retain their specific bindingsites is to be found in Winter & Milstein (1991) Nature 349, 293-299.

By "ScFv molecules" we mean molecules wherein the V_(H) and V_(L)partner domains are linked via a flexible oligopeptide.

It may be advantageous to use antibody fragments, rather than wholeantibodies. Effector functions of whole antibodies, such as complementbinding, are removed. ScFv and dAb antibody fragments can be expressedas fusions with other polypeptides.

Minimal recognition units may be derived from the sequence of one ormore of the complementary-determining regions (CDR) of the Fv fragment.Whole antibodies, and F(ab')₂ fragments are "bivalent". By "bivalent" wemean that the said antibodies and F(ab')₂ fragments have two antigencombining sites. In contrast, Fab, Fv, ScFv, dAb fragments and minimalrecognition units are monovalent, having only one antigen combiningsites.

In a further embodiment the binding moiety is at least part of a ligandof a target cell-specific cell-surface receptor.

It is preferred that the target cell-specific cell-surface receptor isthe receptor for human gonadotrophin releasing hormone (GnRH). In thispreferred embodiment the binding moiety is GnRH, and its bindingspecificity is for human cancer cells that express the GnRH receptors ontheir surface. Examples of such human cancer cells are prostate, breastand endometrial cancer cells.

It is also preferred that the target cell-specific cell-surface receptoris the receptor for melanocyte-stimulating hormone (MSH) which isexpressed in high number in melanoma cells. In this preferred embodimentthe binding moiety is MSH, and its binding specificity is for melanomacells.

It is also preferred that the target cell-specific cell-surface receptoris the receptor for somatostatin.

Of course, the receptors for GnRH, MSH and somatostatin may themselvesbe target cell-specific antigens and may be recognised by bindingmoieties which have the property of any one of a monoclonal antibody, aScFv, a dAb or a minimal recognition unit. Thus, although the bindingsite on the target cell may be a cell-surface receptor it may also actas a target cell-specific cell-surface antigen for recognition by thebinding moiety.

It will be appreciated by those skilled in the art that binding moietieswhich are polypeptides may be conveniently made using recombinant DNAtechniques. The binding moiety may be fused to a protein on the surfaceof the virus or virus-like protein as disclosed below or they may besynthesised independently of the virus or virus-like particle, byexpression from a suitable vector in a suitable host and then joined tothe virus or virus-like particle as disclosed below.

Nucleic acid sequences encoding many of the targeting moieties areknown, for example those for peptide hormones, growth factors, cytokinesand the like and may readily be found by reference to publiclyaccessible nucleotide sequence databases such as EMBL and GenBank. Oncethe nucleotide sequence is known it is obvious to the person skilled inthe art how to make DNA encoding the chosen binding moiety using, forexample, chemical DNA synthetic techniques or by using the polymerasechain reaction to amplify the required DNA from genomic DNA or fromtissue-specific cDNA.

Many cDNAs encoding peptide hormones, growth factors, cytokines and thelike, all of which may be useful as binding moieties, are generallyavailable from, for example British Biotechnology Ltd, Oxford, UK.

It is preferred that when the virus or virus-like particle of theinvention binds to its target cell it delivers its nucleic acid to thesaid target cell, that is the target cell is infected by the virus orvirus-like particle. Target cells, especially cancer cells, that areinfected in this manner by the virus or virus-like particle may expressviral molecules on their surface and may be recognised by the immunesystem and destroyed. Of course, other cytotoxic functions of the virusmay also kill the cell.

In one embodiment when the virus or virus-like particle is adenovirus,the E1B gene is substantially deleted or modified so that its geneproduct no longer interacts with the E1A protein. E1A protein stimulatesapoptosis but normally its action is inhibited by E1B. Conveniently, theE1B gene is inactivated by insertion; preferably a cytotoxic gene, asdefined below, is inserted at or near the E1B gene.

E1, E3 and a site upstream of E4 may be used as sites for insertion offoreign DNA sequences in the generation of recombinant adenoviruses forexample see Berkner and Sharp (1984) Nucl. Acids Res. 12, 1925-1941;Chanda et al (1990) Virology 175, 535-547; Haj-Ahmad and Graham (1986)J. Virol. 57, 267-274; Saito et al (1985) J. Virol. 54, 711-719; allincorporated herein by reference. Since the upper size limit for DNAmolecules that can be packaged into adenovirus particles isapproximately 105% of the wild-type genome only about 2 kb of extra DNAcan be inserted without compensating deletions of viral DNA. Although E1is essential for virus replication in cell culture, foreign DNA can besubstituted for E1 sequences when the virus is grown in 293 cells whichare transformed by Ad5 DNA and constitutively express E1 (Graham et al(1977) J. Gen. Virol. 36, 59-72, incorporated herein by reference).Several vectors having 1.9 kb deleted from E3 of Ad5 have beenconstructed without interfering with virus replication in cell culture(reviewed by Graham and Prevec (1992) in "Vaccines: New Approaches toImmunological Problems" R. W. Ellis (Ed.), Butterworth-Heinemann,Boston, Mass., pages 364-390, incorporated herein by reference). Suchvectors allow for insertion of up to 4 kb of foreign DNA. Recombinantadenoviruses containing inserts in E3 replicate in all Ad-permissivecell lines and a number of adenovirus vectors containing E3 inserts havebeen shown to express foreign genes efficiently both in vitro and invivo (Berkner (1988) Biotechniques 6, 616-629; Chanda et al (1990)Virology 175, 535-547; Dewar et al (1989) J. Virol. 63, 129-136; Graham(1990) Trends Biotechnol. 8, 85-87; Graham and Prevec (1992) in"Vaccines: New Approaches to Immunological Problems" R. W. Ellis (Ed.),Butterworth-Heinemann, Boston, Mass., pages 364-390; Johnson et al(1988) Virology 164, 1-14; Lubeck et al (1989) Proc. Natl. Acad. Sci.USA 86, 6763-6767; McDermott et al (1989) Virology 169, 244-247; Morinet al (1987) Proc. Natl. Acad. Sci. USA 84, 4626-4630; Prevec et al(1989) J. Gen. Virol. 70, 429-434; Prevec et al (1990) J. Inf. Dis. 161,27-30; Schneider et al (1989) J. Gen. Virol. 70, 417-427; Vernon et al(1991) J. Gen. Virol. 72, 1243-1251; Yuasa et al (1991) J. Gen. Virol.72, 1927-1934) all incorporated herein by reference.

Substantially replication-defective adenoviruses may be made by creatinga deficiency of the E1A protein. Suitably this is achieved by deletingthe E1A gene or by making mutations within the E1A gene that preventexpression of the E1A protein. Examples of suitable mutations aredeletions within the E1A coding region; nonsense mutations; andframeshift mutations.

In further preference, the virus or virus-like particle is modifiedfurther to contain a gene suitable for gene therapy.

In one embodiment, the gene encodes a molecule having a directly orindirectly cytotoxic function. By "directly or indirectly" cytotoxic, wemean that the molecule encoded by the gene may itself be toxic (forexample ricin; tumour necrosis factor; interleukin-2; interferon-gamma;ribonuclease; deoxyribonuclease; Pseudomonas exotoxin A) or it may bemetabolised to form a toxic product, or it may act on something else toform a toxic product. The sequence of ricin cDNA is disclosed in Lamb etal (1985) Eur. J. Biochem. 148, 265-270 incorporated herein byreference.

For example, it would be desirable to target a DNA sequence encoding anenzyme using the virus or virus-like particle of the invention, theenzyme being one that converts a relatively non-toxic prodrug to a toxicdrug. The enzyme cytosine deaminase converts 5-fluorocytosine (5FC) to5-fluorouracil (5FU) (Mullen et al (1922) PNAS 89, 33); the herpessimplex enzyme thymidine kinase sensitises cells to treatment with theantiviral agent ganciclovir (GCV) or aciclovir (Moolten (1986) CancerRes. 46, 5276; Ezzedine et al (1991) New Biol 3, 608). The cytosinedeaminase of any organism, for example E. coli or Saccharomycescerevisiae, may be used.

Thus, in a preferred embodiment of the invention, the gene encodes acytosine deaminase and the patient is concomitantly given 5FC. By"concomitantly", we mean that the 5FC is administered at such a time, inrelation to the transformation of the tumour cells, that 5FC isconverted into 5FU in the target cells by the cytosine deaminaseexpressed from the said gene. A dosage of approximately 0.001 to 100.0mg 5C/kg body weight/day, preferably 0.1 to 10.0 mg/kg/day is suitable.

Components, such as 5FC, which are converted from a relatively non-toxicform into a cytotoxic form by the action of an enzyme are termed"pro-drugs".

Other examples of pro-drug/enzyme combinations include those disclosedby Bagshawe et al (WO 88/07378), namely various alkylating agents andthe Pseudomonas spp. CPG2 enzyme, and those disclosed by Epenetos &Rowlinson-Busza (WO 91/11201), namely cyanogenic pro-drugs (for exampleamygdalin) and plant-derived β-glucosidases.

Enzymes that are useful in this embodiment of the invention include, butare not limited to, alkaline phosphatase useful for convertingphosphate-containing prodrugs into free drugs; arylsulfatase useful forconverting sulfate-containing prodrugs into free drugs; cytosinedeaminase useful for converting non-toxic 5-fluorocytosine into theanti-cancer drug, 5-fluorouracil; proteases, such as serratia protease,thermolysin, subtilisin, carboxypeptidases and cathepsins (such ascathepsins B and L), that are useful for converting peptide-containingprodrugs into free drugs; D-alanylcarboxypeptidases, useful forconverting prodrugs that contain D-amino acid substituents;carbohydrate-cleaving enzymes such as β-galactosidase and neuraminidaseuseful for converting glycosylated prodrugs into free drugs; β-lactamaseuseful for converting drugs derivatized with β-lactams into free drugs;and penicillin amidases, such as penicillin V amidase or penicillin Gamidase, useful for converting drugs derivatized at their aminenitrogens with phenoxyacetyl or phenylacetyl groups, respectively, intofree drugs. Alternatively, antibodies with enzymatic activity, alsoknown in the art as abzymes, can be used to convert the prodrugs of theinvention into free active drugs see, e.g. R. J. Massey, Nature, 328,pp. 457-458 (1987)!.

Similarly, the prodrugs of this invention include, but are not limitedto, the above-listed prodrugs, e.g., phosphate-containing prodrugs,thiophosphate-containing prodrugs, sulfate-containing prodrugs,peptide-containing prodrugs, D-amino acid-modified prodrugs,glycosylated prodrugs, β-lactam-containing prodrugs, optionallysubstituted phenoxyacetamide-containing prodrugs or optionallysubstituted phenylacetamide-containing prodrugs, 5-fluorocytosine andother 5-fluorouridine prodrugs which can be converted by the enzyme ofthe conjugate into the more active, cytotoxic free drug. Examples ofcytotoxic drugs that can be derivatized into a prodrug form for use inthis invention include, but are not limited to, etoposide, teniposide,adriamycin, daunomycin, carminomycin, aminopterin, dactinomycin,mitomycins, cis-platinum and cis-platinum analogues, bleomycins,esperamicins see U.S. Pat. No. 4,675,187!, 5-fluorouracil, melphalan andother related nitrogen mustards.

In a further embodiment the gene delivered to the target cell encodes aribozyme capable of cleaving targeted RNA or DNA. The targeted RNA orDNA to be cleaved may be RNA or DNA which is essential to the functionof the cell and cleavage thereof results in cell death or the RNA or DNAto be cleaved may be RNA or DNA which encodes an undesirable protein,for example an oncogene product, and cleavage of this RNA or DNA mayprevent the cell from becoming cancerous.

Ribozymes which may be encoded in the genomes of the viruses orvirus-like particles herein disclosed are described in Cech andHerschlag "Site-specific cleavage of single stranded DNA" U.S. Pat. No.5,180,818; Altman et al "Cleavage of targeted RNA by RNAse P" U.S. Pat.No. 5,168,053, Cantin et al "Ribozyme cleavage of HIV-1 RNA" U.S. Pat.No. 5,149,796; Cech et al "RNA ribozyme restriction endoribonucleasesand methods", U.S. Pat. No. 5,116,742; Been et al "RNA ribozymepolymerases, dephosphorylases, restriction endonucleases and methods,U.S. Pat. No. 5,093,246; and Been et al "RNA ribozyme polymerases,dephosphorylases, restriction endoribonucleases and methods; cleavessingle-stranded RNA at specific site by transesterification", U.S. Pat.No. 4,987,071, all incorporated herein by reference.

In a still further embodiment the gene delivered to the target cellencodes an antisense RNA.

By "antisense RNA" we mean an RNA molecule which hybridises to, andinterferes with the expression from a mRNA molecule encoding a proteinor to another RNA molecule within the cell such as pre-mRNA or tRNA orrRNA, or hybridises to, and interferes with the expression from a gene.

Conveniently, a gene expressing an antisense RNA may be constructed byinserting a coding sequence encoding a protein adjacent a promoter inthe appropriate orientation such that the RNA complementary to mRNA.Suitably, the antisense RNA blocks expression of undesirablepolypeptides such as oncogenes, for example ras, bcl, src or tumoursuppressor genes such as p53 and Rb.

It will be appreciated that it may be sufficient to reduce expression ofthe undesirable polypeptide rather than abolish the expression.

It will be further appreciated that DNA sequences suitable forexpressing as antisense RNA may be readily derived from publiclyaccessible databases such as GenBank and EMBL.

In another embodiment of the invention, the gene replaces the functionof a defective gene in the target cell.

There are several thousand inherited genetic diseases of mammals,including humans, that are caused by defective genes. Examples of suchgenetic diseases include cystic fibrosis, where there is known to be amutation in the CFTR gene; Duchenne muscular dystrophy, where there isknown to be a mutation in the dystrophin gene; sickle cell disease,where there is known to be a mutation in the HbA gene. Many types ofcancer are caused by defective genes, especially protooncogenes, andtumour-suppressor genes that have undergone mutation.

Thus, it is preferred that the virus or virus-like particle of theinvention, which may be useful in the treatment of cystic fibrosis,contains a functional CFTR gene to replace the function of the defectiveCFTR gene. Similarly, it is preferred that the virus or virus-likeparticle of the invention, which may be useful in the treatment ofcancer, contains a functional protooncogene, or tumour-suppressor geneto replace the function of the defective protooncogene ortumour-suppressor gene.

Examples of protooncogenes are ras, src, bcl and so on; examples oftumour-suppressor genes are p53 and Rb.

By "gene" we mean a nucleic acid coding sequence that may containintrons, or fragment thereof, or cDNA, or fragment thereof.

It will be appreciated that the gene will be introduced into aconvenient place within the genome of the virus or virus-like particleand will contain a promoter and/or enhancer element to drive itsexpression.

It is preferred if the promoter and/or enhancer is selective for thecells to be targeted. Some examples of tissue or tumour specificpromoters are given below but new ones are being discovered all of thetime which will be useful in this embodiment of the invention.

The tyrosinase and TRP-1 genes both encode proteins which play key rolesin the synthesis of the pigment melanin, a specific product ofmelanocytic cells. The 5' ends of the tyrosinase and tyrosinase-relatedprotein (TRP-1) genes confer tissue specificity of expression on genescloned downstream of these promoter elements.

The 5' sequences of these genes are described in Bradl, M. et al (1991)Proc. Natl. Acad. Sci. USA 88, 164-168 and Jackson, I. J. et al (1991)Nucleic Acids Res. 19, 3799-3804.

Prostate-specific antigen (PSA) is one of the major protein constituentsof the human prostate secretion. It has become a useful marker for thedetection and monitoring of prostate cancer. The gene encoding PSA andits promoter region which directs the prostate-specific expression ofPSA have been described (Lundwall (1989) Biochem. Biophys. Res. Comm.161, 1151-1159; Riegman et al (1989) Biochem. Biophys. Res. Comm. 159,95-102; Brawer (1991) Acta Oncol. 30, 161-168).

Carcinoembryonic antigen (CEA) is a widely used tumour marker,especially in the surveillance of colonic cancer patients. Although CEAis also present in some normal tissues, it is apparently expressed athigher levels in tumorous tissues than in corresponding normal tissues.The complete gene encoding CEA has been cloned and its promoter regionanalysed. A CEA gene promoter construct, containing approximately 400nucleotides upstream from the translational start, showed nine timeshigher activity in the adenocarcinoma cell line SW303, compared with theHeLa cell line. This indicates that cis-acting sequences which conveycell type specific expression are contained within this region (Schreweet al (1990) Mol. Cell. Biol. 10, 2738-2748).

The c-erbB-2 gene and promoter have been characterised previously andthe gene product has been shown to be over-expressed in tumour celllines (Kraus et al (1987) EMBO J. 6, 605-610).

The mucin gene, MUC1, contains 5' flanking sequences which are able todirect expression selectively in breast and pancreatic cell lines, butnot in non-epithelial cell lines as taught in WO 91/09867.

The binding moiety allowing the virus or virus-like particle to bind toa target cell may be a polypeptide or oligosaccharide or lipid or anyother molecule capable of binding specifically to the target cell.

It is preferred that the binding moiety is a polypeptide.

The molecule on the surface of the virus or virus-like particle to whichthe binding moiety is joined may be a polypeptide, oligosaccharide orlipid or any other molecule in the virus or virus-like particle coat. Itis preferred that the molecule is a polypeptide.

If the binding moiety and the molecule on the surface of the virus orvirus-like particle are both polypeptides then they may be linkedtogether by any of the conventional ways of cross-linking polypeptides,such as those generally described in O'Sullivan et al Anal. Biochem.(1979) 100, 100-108. For example, the binding moiety may be enrichedwith thiol groups and the molecule on the surface of the virus orvirus-like particle reacted with a bifunctional agent capable ofreacting with those thiol groups, for example the N-hydroxysuccinimideester of iodoacetic acid (NHIA) orN-succinimidyl-3-(2-pyridyldithio)propionate (SPDP). Amide and thioetherbonds, for example achieved with m-maleimidobenzoyl-N-hydroxysuccinimideester, are generally more stable in vivo than disulphide bonds.

Other chemical procedures may be useful in joining oligosaccharide andlipids to other oligosaccharides, lipids or polypeptides.

It is preferred that the binding moiety and the molecule on the surfaceof the virus or virus-like particle are both polypeptides that may beproduced as a fusion by the techniques of genetic engineering. The useof genetic engineering allows for the precise control over the fusion ofsuch polypeptides.

Thus a further embodiment of the invention is a nucleotide sequenceencoding the fusion of the binding moiety and the protein on the surfaceof the virus or virus-like particle.

The nucleotide sequence encoding the fusion of the binding moiety andthe protein on the surface of the virus or virus-like particle ispreferably made by an alteration of the viral genome.

The nucleotide sequence may be synthesised de novo using solid phasephosphoramidite chemistry, but it is more usual for the nucleotidesequence to be constructed from two parts, the first encoding thebinding moiety and the second the protein on the surface of the virus orvirus-like particle. The two parts may be derived from their respectivegenes by restriction endonuclease digestion or by other methods known bythose skilled in the art such as the polymerase chain reaction.

A variety of methods have been developed to operatively link twonucleotide sequences via complementary cohesive termini. For instance,synthetic linkers containing one or more restriction sites provide amethod of joining the two DNA segment together. Each DNA segment,generated by endonuclease restriction digestion, is treated withbacteriophage T4 DNA polymerase of E. coli DNA polymerase I, enzymesthat remove protruding, 3'-single-stranded termini with their3'-5'-exonucleolytic activities, and fill in recessed 3'-ends with theirpolymerizing activities.

The combination of these activities therefore generates blunt-ended DNAsegments. The blunt-ended segments are then incubated with a large molarexcess of linker molecules in the presence of an enzyme that is able tocatalyze the ligation of blunt-ended DNA molecules, such asbacteriophage T4 DNA ligase. Thus, the products of the reaction are DNAsegments carrying polymeric linker sequences at their ends. These DNAsegments arc then cleaved with the appropriate restriction enzyme andlifted to an expression vector that has been cleaved with an enzyme thatproduces termini compatible with those of the DNA segment.

Synthetic linkers containing a variety of restriction endonuclease sitesare commercially available from a number of sources includingInternational Biotechnologies Inc, New Haven, Conn., USA.

A desirable way to generate the DNA encoding the fusion polypeptide ofthe invention is to use the polymerase chain reaction as disclosed bySaiki et al (1988) Science 239, 487-491.

In this method each of the DNA molecules encoding the two polypeptidesto be fused are enzymatically amplified using two specificoligonucleotide primers which themselves become incorporated into theamplified DNA. The said specific primers may contain restrictionendonuclease recognition sites which may then be used to join the saidtwo DNA molecules using T4 DNA ligase as disclosed.

A particular feature of one aspect of the present invention is themodification of the virus or virus-like particle of the invention sothat it no longer binds its host cell and so that it binds the targetcell by virtue of its binding moiety.

The host-cell receptor of adenovirus may be the penton fibre and that ofinfluenza virus may be the haemagglutinin receptor.

These receptors may be modified by the insertion or deletion orsubstitution of amino acid residues that disrupt their host-cell bindingfunction. It is preferred that the binding moiety for the target cell isjoined to the host-cell receptor in such a manner that the bindingmoiety is capable of binding the target cell, the host-cell receptor isunable to bind to the host cell and therefore the binding specificity ofthe virus or virus-like particle is modified. A further preference isthat the portion of the host-cell receptor that is exposed on thesurface of the virus or virus-like particle is replaced by the bindingmoiety, and that the portion of the host-cell receptor which promotesthe uptake of viral DNA by the target cell is retained. Suitably, thebinding moiety is joined directly or indirectly to the host-cellreceptor by a spacer group.

Examples of spacer groups are polypeptide sequences of between 4 and1000 amino acid residues.

Thus, in one embodiment of the invention the gene encoding the pentonfibre in adenovirus is modified in such a way that the DNA encoding thesurface-exposed portion is replaced by a DNA fragment encoding a ScFv,the ScFv being derived from an antibody which binds to a target cellsurface antigen.

Potential fusion sites within the penton fibre have been identified.

The adenovirus fibre is a trimer composed of three protomers. The aminoterminal end (40 amino acids or so) of each participates in theformation of a tail that is closely associated with the penton (asopposed to the hexon) subunit of the capsid. High amino acidconservation is maintained between the different characterisedserotypes.

Middle portions of each protomer form the shaft of the protein. Thisshaft is of variable length, depending upon serotype, and is composed ofrepeating units of 15 amino acids (for examples, serotypes have beenidentified with 6, 15 and 21 repeat units). These repeating units arenot duplicates: rather than strict conservation of amino acid structure,there is a general conservation of relative hydrophobicity. Someserotypes, for example, 40 and 41, have shafts composed of differentlength fibre proteins. This suggests a certain flexibility in structuralconstraints.

The carboxy-terminal ends (some 200 amino acids) associate to form aknob that is held erect a great distance (in molecular terms) from thecapsid.

Whilst the cellular receptor(s) and mechanisms of docking have not beenfirmly identified and elucidated, we propose that the most likelycandidate structure for cell binding is the knob. Thus, in oneembodiment the whole knob of the penton fibre has been replaced withsingle chain antibody (ScFv) domains. The triplex structure implies thateach fibre will thus end in three ScFvs. Additionally, the ScFv regionscan be replaced with CDRs, or by non-antibody derived peptides, of knownspecificity or other molecules that are capable of interactingspecifically with the target cell.

Suitable fusion sites are therefore at the native junction between shaftand knob domains, or (should the DNA sequence prove to be more amenable)at any junction between repetitive units of the shaft. Preferably, theminimum shaft length is not reduced beyond the smallest size naturallyidentified. There are thus at least 15 potential sites at which fusioncould be contemplated.

Although it is preferred that the binding moiety forms the end of thefibre thereby replacing the knob, the binding moiety may also be fusedwithin the penton fibre sequence but still display its binding surfacesand bind to the target cell.

Suitably, the binding moiety may be fused to the knob and extendexternally to the knob structure.

In a further embodiment influenza virus haemmaglutinin is modified toincorporate a binding moiety. Influenza virus has seven or eight(depending on serotype) genetic segments, all negative strand RNA.Suitably, a cDNA from the whole segment encoding haemmagglutinin isconstructed and modified by adding a promoter firing backwards acrossthis segment so that negative strand RNA is made. Genetic fusions with asuitable binding molecule, as disclosed above, are made using standardrecombinant DNA methods and a suitable cell line is stably transfectedwith this gene construct. Infection of this transfected cell line withinfluenza virus and selection of reassorted genomes containing the newhaemmagglutinin by infection of a normally resistant cell line thatexpresses a marker that can only be recognised by the newhaemmagglutinin yields the desired virus comprising modifiedcell-binding specificity.

A further aspect of the invention provides a method of producing in cellculture a virus or virus-like particle and then joining the bindingmoiety, as defined above, to the virus or virus-like particle.

A further aspect of the invention provides a method of producing in cellculture a virus or virus-like particle which has been geneticallymodified to express a binding moiety on its surface. The virus orvirus-like particle is grown in its host prior to modification, but oncethe modification that alters the binding specificity is made, the virusor virus-like particle is grown in the target cell. Thus, for example inthe case where the binding moiety recognises a breast tumour cellantigen, the virus or virus-like particle is grown in breast tumour cellculture.

The virus or virus-like particles of the invention are administered inany suitable way, usually parenterally, for example intravenously,intraperitoneally or intravesically, in standard sterile, non-pyrogenicformulations of diluents and carriers, for example isotonic saline (whenadministered intravenously).

A further aspect of the invention provides a method of delivery of thevirus or virus-like particle which contains a gene encoding a moleculehaving an indirectly cytotoxic function.

Suitably, the indirectly cytotoxic function is an enzyme that converts aprodrug to a toxic drug. With such a virus or virus-like particle, oncethe virus or virus-like particle has bound to the target cells,delivered its nucleic acid to the cells, and expressed the indirectlycytotoxic functions, which typically takes a day or so, the pro-drug isadministered. The timing between administration of the virus orvirus-like particle and the pro-drug may be optimised in a non-inventiveway.

The dosage of the pro-drug will be chosen by the physician according tothe usual criteria. The dosage of the virus or virus-like particle willsimilarly be chosen according to normal criteria, and in the case oftumour treatment, particularly with reference to the type, stage andlocation of tumour and the weight of the patient. The duration oftreatment will depend in part upon the rapidity and extent of any immunereaction to the virus or virus-like particle.

Some of the viruses or virus-like particles either in themselves, ortogether with an appropriate pro-drug, are in principle suitable for thedestruction of cells in any tumour or other defined class of cellsselectively exhibiting a recognisable (surface) entity. Examples oftypes of cancer that may be treated using the viruses or virus-likeparticles are cancer of the breast, prostate, colon, rectum, ovary,testicle and brain. The compounds are principally intended for human usebut could be used for treating other mammals including dogs, cats,cattle, horses, pigs and sheep.

The invention will now be described in detail with reference to thefollowing Figures and Examples in which:

FIG. 1(a)-(c) shows (a) an unmodified (i.e. "naive") virus or virus-likeparticle able to bind to and infect its host cell but not a non-hostcell, such as a target cell; and (b) a virus or virus-like particle witha modified binding specificity does not bind and infect its host cellbut binds and infects a target cell; and (c) a virus or virus-likeparticle as in (b) modified further to contain a gene for gene therapyor cancer treatment.

FIG. 2(a)-(c) shows (a) unmodified (naive) adenovirus; (b) adenovirusmodified so that its penton fibres, which recognise the host cell, arereplaced in part by antibody fragments which recognise the target cell;and (c) adenovirus as in (b) with further genetic material added to theviral DNA for gene therapy of cancer.

FIG. 3(a)-(b) shows (a) influenza virus and (b) genetically-modifiedinfluenza virus wherein at least part of the haemagglutinin binding siteis replaced by an antibody with anti-cancer cell binding activity.

FIG. 4(a)-(b) shows (a) a retrovirus virus; and (b) as in (a) except theretrovirus has been modified further to express on its surface ananticancer cell-binding antibody fragment or an anticancer cell-bindingpeptide.

FIG. 5 is a diagrammatic representation of a penton fibre indicatingpotential fusion sites within the fibre.

FIG. 6 shows fusions between the DNA encoding the Ad5 fibre and an ScFv(SEQ. ID NO. 1 through 43, as depicted in the Key to Sequence Listingset forth hereinafter.

FIG. 7 shows sequences of oligonucleotides used for amplifying the ScFv.All oligonucleotides are presented 5' to 3', the reverse complement ofFOR primers are shown and derived amino acid sequences are shown whererelevant. (SEQ. ID NO. 46 and 47, as depicted in the Key to SequenceListing set forth hereinafter).

FIG. 8 shows the construction of plasmid pRAS117.

FIG. 9 shows the nucleotide and derived amino acid sequence between theHindIII and EcoRI sites of pRAS117. (SEQ. ID NO. 49, as depicted in theKey to Sequence Listing set forth hereinafter).

FIG. 10 shows a map of plasmid pRAS117.

FIG. 11 is a diagrammatic representation of the construction of plasmidpRAS118.

FIG. 12 shows the sequences of oligonucleotides for amplifying Ad5 fibreDNA fragments. All oligonucleotides are presented 5'→3'. The reversecomplements of FOR primers are shown. Derived amino acid sequences areshown where relevant. (SEQ. ID No. 51 through 69, as depicted in the Keyto Sequence Listing set forth hereinafter).

FIG. 13 shows the nucleotide sequence and deduced amino acid sequencebetween the HindIII site and EcoRI site of pRAS111. (SEQ ID No. 71, asdepicted in the Key to Sequence Listing set forth hereinafter).

FIG. 14 gives a diagrammatic representation of constructing adenoviruscarrying a cytotoxic gene.

FIG. 15(a) gives the nucleotide and amino acid sequences of mouse andhumanised HMFG1 variable regions. (SEQ ID No. 73 as depicted in the Keyto Sequence Listing set forth hereinafter).

FIG. 15(b) gives the nucleotide and amino acid sequences of mouse andhumanised HMFG1 variable regions (SEQ ID No. 75).

EXAMPLE 1

Fusion sites within the adenovirus Ad5 fibre for binding moietiesincluding single chain Fv (ScFv)

The Ad5 DNA sequence co-ordinates used here are taken from:

ADRCOMPGE₋₋ 1: residues 1 to 32760

and

ADRCOMPGE₋₋ 2: residues 32761-35935

These can be accessed by using program SEQ on the Intelligeneticsdatabase.

The sequence of Ad5 fibre can also be found in Chroboczek, J. andJacrot, B. (1987) "The sequence of adenovirus fiber: Similarities anddifferences between serotypes 2 and 5" Virology 161, 549-554 and isavailable from the EMBL Database, Heidelberg, Germany under accessionname ADEFIB.

Fusion sequences between the shaft and the ScFv are shown in FIG. 6. Thefusion sites are at the junctions of the repetitive units of the shaft.Shaft sequences are shown in normal typescript; ScFv sequences are shownin italics. The DNA sequence between the PstI and XhoI sites is uniqueto the ScFv used.

Fusion A is at the end of the first repetitive unit of the shaft(co-ordinates 31218-9), fusion B at the end of the second (31266-7),fusion C at the third (31323-4), fusion D at the fourth (31368-9),fusion E at the fifth (31413-4), fusion F at the sixth (31458-9), fusionG at the seventh (31503-4), fusion H at the eighth (31551-2), fusion Iat the ninth (31596-7), fusion J at the tenth (31641-2), fusion K at theeleventh (31692-3), fusion L at the twelfth (31737-8), fusion M at thethirteenth (31787-8), fusion N at the fourteenth (31836-7), fusion O atthe fifteenth (31884-5), fusion P at the sixteenth (31929-30), fusion Qat the seventeenth (31995-6), fusion R at the eighteenth (32040-1),fusion S at the nineteenth (32103-4), fusion T at the twentieth(32151-2), fusion U at the twenty-first (32199-200), and fusion V is atthe end of the twenty-second repetitive unit of the shaft (32244-5), thejunction between shaft and knob.

EXAMPLE 2

Preparation of adenovirus expressing an ScFv on its surface

The genetically modified fibre is introduced into the Ad5 genome by: (a)replacing the fibre gene of plasmid pE4 with the modified fibre bystandard recombinant DNA technology and (b) reconstituting the virus byrecombination.

pE4 is a plasmid containing the right hand half of the Ad5 genome, andwhich has served as the source of the Ad5 fibre gene that we have used.It was provided by Dr Keith Leppard, Biological Sciences, University ofWarwick, Coventry, CV4 7AL who has supplied details of its structure. Ifit is introduced into mammalian cells that contain the remainder of theAd5 genome, then it is possible to obtain recombinants containing themodification. Most human cell lines can be used for the recombinationbut HeLa cells are preferred.

The plasmid pE4 is readily made in the following way. A derivative ofpBR322 is made by digesting with BstN1 and rejoining using XhoI linkerssuch that the BstN1 fragment corresponding to positions 1442-2502 in thepBR322 sequence is removed. DNA from the adenovirus Ad5 strain 309described by Jones & Shenk (1979) Cell 17, 683-689 is isolated anddeproteinated. This DNA is then ligated to ClaI linkers and cut withEcoRI and ClaI. The ClaI-EcoRI fragment corresponding to the region of76% of the Ad5 genome to the right hand end is isolated and cloned intothe EcoRI-ClaI sites of the above-mentioned pBR322 derivative to formpE4.

Adenovirus Type 5 and HeLa cells are available from the American TypeCulture Collection, 10801 University Blvd., Manassas, Va. 20110-2209,USA under accession numbers ATCC VR-5 and ATCC CCL-2.

Construction of plasmid pRAS117

Oligonucleotide primers LEADHBACK and LEADbFOR (FIG. 7) were used forPCR-mediated amplification of the DNA segment extending from the HindIIIsite of plasmid pRAS111, over the Shine-Dalgarno sequence and the pelBleader sequence to the PstI site in the ScFv. LEADbFOR directs theincorporation of a BglII site immediately after the pelB leadersequence. DNA (100 ng) from plasmid pRAS111 was subjected to 24 roundsof amplification, (94° C., 1 min; 65° C., 1.5 min and 72° C., 2 min) ina 50 μl reaction volume containing 25 pmol of each primer, 250 mM ofeach dNTP, 67 mM Tris-HCl (pH 8.8), 17 mM (NH₄)₂ SO₄, 1.5 mM MgCl₂, 200mg.ml⁻¹ gelatine and 5 units of Thermus aquaticus (Taq) polymerase(Cetus) overlaid with 25 μl paraffin oil. After the reaction, oil wasremoved by extraction with 500 μl chloroform. The sample was loaded on a2% agarose gel, and the amplified fragment was electrophoresed on to apiece of NA45 paper (Schleicher and Schuell). Bound DNA was subsequentlyeluted by immersion in 400 μl 1M NaCl made in TE (10 mM Tris-HCl, pH7.5, 1 mM EDTA) for 30 min at 70° C. To this was added 800 μl ethanol,and after incubation (2 h, -20° C.) the DNA was collected bycentrifugation. The pellet was taken up in 50 μl T/E.

One fifth (10 μl) of the purified amplified fragment was cut with therestriction enzymes HindIII and PstI, in a total volume of 20 μl 50 mMTris-HCl, pH 7.5, 10 MgCl2, 100 mM NaCl, 1 mM dithioerythreitolcontaining 10 units of each enzyme. After incubation (1 h, 37° C.) thereaction was stopped by incubation at 70° C. for 15 minutes.

The trimmed amplified fragment was cloned between the HindIII and PstIsites of pUC8, to generate plasmid pRAS117.

Plasmid pUC8 (1 μg) was cut with HindIII and PstI, in a total volume of20 μl 50 mM Tris-HCl, pH 7.5, 10 MgCl₂, 100 mM NaCl, 1 mMdithioerythreitol containing 10 units of each enzyme. After incubation(1 h, 37° C.) the reaction was stopped by incubation at 70° C. for 15minutes.

The ligation reaction contained 1.5 μl of pUC8/HindIII, PstI and 3 μl ofthe amplified leader/HindIII, PstI in a total volume of 15 μl containing70 mM Tris-HCl pH 7.5, 7 mM MgCl₂, 0.7 mM rATP, 4 mM dithiothreitol, 0.5mg.ml⁻¹ BSA and 10 units of T4 DNA ligase. After incubation (2 h, atroom temperature), the reaction was stopped by the addition of 1 μl 500mM EDTA, pH 8.0 and 14 μl H₂ O.

This ligation mix was used to transform E. coli.

An aliquot (5 μl) of this ligation mix was used to transform a 200 μlaliquot of commercially available competent E. coli K12 DH58,1αF (LifeSciences Inc). After incubation (30 min, 0° C.), heat shock (2 min, 42°C.), addition of 800 μl L-broth and recovery (37° C., 1 h), cells (100μl) were spread on L-agar plates containing 100 μg.ml⁻¹ ampicillincontaining 50 mM IPTG (isopropyl-β-D-galactopyranoside) and 100 μg.ml⁻¹X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside). Cells weregrown overnight at 37° C., and individual colonies were transferred tofresh L-agar/ampicillin plates. After 6 h growth, colonies were used toinoculate 5 ml aliquots of L-broth containing 100 μg.ml³¹ 1 ampicillin.These cells were grown overnight with shaking at 37° C., and used as asource of plasmid DNA.

These cells were used as a source of plasmid DNA.

Harvested cells were suspended in 360 μl of SET (50 mM sucrose, 10 mMEDTA, 100 mM Tris-HCl, pH 7.5) containing 2 mg.ml⁻¹ hen egg. lysozyme,transferred to a 1.5 ml microfuge tube, and diluted by addition of 300μl 10% Triton X-100. After floating on boiling water for 2 min andcooling for a further minute in ice/water, denatured cell debris wasremoved by centrifugation (14,000×g, 20 min) in a microcentrifuge. Themajority of the soluble remaining proteins were removed by addition of300 μl 7.5M ammonium acetate and centrifugation (14,000×g, 10 min).Nucleic acids were precipitated by addition of 720 μl cold (-20° C.)isopropanol and centrifugation (14,000×g, 10 min). After rinsing thepellets with ethanol and drying, DNA was solubilised in 60 μl TEcontaining 170 μg.ml⁻¹ RNase A.

Restriction enzyme digestions on 5 μl aliquots, using the enzymesHindIII and BglII identified which of these plasmids were pRAS117. Theconstruction scheme is shown in FIG. 8. The nucleotide and derived aminoacid sequences between the HindIII and EcoRI sites of pRAS117 are shownin FIG. 9. A map of plasmid pRAS117 is provided in FIG. 10.

The nucleotide sequence of the relevant portion of pRAS111, between theHindIII site and EcoRI, site is given in FIG. 13.

Construction of plasmid pRAS118 (FIG. 11)

The 130 bp HindIII-PstI fragment of pRAS117 was used to replace thecorresponding fragment of pRAS111, to generate plasmid pRAS118. Analiquot (2 μg) of pRAS111 DNA was cut with HindIII and PstI in theconditions used previously, the large fragment was isolated byelectrophoresis onto NA45 paper, as described previously, and the DNAwas suspended in 10 μl of TE. An aliquot (10 μl) of pRAS117 DNA was cutwith HindIII and PstI in the conditions used previously, and the smallfragment was isolated by electrophoresis onto NA45 paper, as describedpreviously, and the DNA was suspended in 10 μl of TE.

The isolated pRAS111/HindIIIPstI large fragment (1.5 μl) and theisolated pRAS117HindIIIPstI small fragment (3 μl) were mixed and ligatedin the conditions previously described.

Transformations, colony handling and DNA preparations were as previouslydescribed.

Restriction enzyme digestions on 5 μl aliquots, using the enzymesHindIII, PstI and BglII identified which of these plasmids were pRAS118.This encodes a NIP-reactive ScFv with a BglII cloning site immediatelydownstream of the pelb leader, suitable for inserting fragments of DNAfrom Ad5 fibre (and also suitable for fusion of any other desired fusionfunctions).

Amplification of Ad5 fibre DNA fragments

Fragments of DNA from Ad5 fibre were amplified by PCR usingoligonucleotide TAILbBACK and oligonucleotide FIBREPFOR, FIBRE3FOR,FIBRE6FOR, FIBRE9FOR, FIBRE12FOR, FIBRE15FOR, FIBRE18FOR, FIBRE21FOR orFIBRE22FOR. Oligonucleotide sequences can be found in FIG. 12.

TAILdBACK directs the incorporation of a BglII site at the base of thefibre, and the FIBREnFOR series primers direct the incorporation of aPstI site at the junctions of repetitive shaft units 3-4 (FIBRE3FOR),6-7 (FIBRE6FOR), 9-10 (FIBRE9FOR), 12-13 (FIBRE12FOR), 15-16(FIBRE15FOR), 18-19 (FIBRE18FOR), 21-22 (FIBRE21FOR), between unit 22and the knob (FIBRE22FOR) or at the end of the knob sequence(FIBREPFOR).

Fusion of fibre and ScFv

The amplified segments of fibre are trimmed with BglII and PstI andligated between the BglII and PstI sites of plasmid pRAS118. This givesa range of fusions under the transcriptional control of the T7 promoter.Colonies are recovered after transformation of a suitable E. colistrain, such as DH5, which does not permit expression of the fusions.

Screening

Colonies containing candidates for fusion are identified by restrictiondigestion of their plasmid DNAs. These candidate DNAs are used totransform a suitable E. coli strain, such as BL21 (DE3), that contains achromosomal insertion of T7 polymerase under lac control. In thesecells, induction of expression of T7 polymerase using the gratuitousinducer IPTG causes expression of the fusion proteins. SolubleNIP-reactive material is identified in colonies with correctly assembledfusions. The DNA of these is identified and the NIP-reactive ScFvderived from pRAS111 are replaced with a cell-binding ScFv.

Replacing the fibre:ScFv in plasmid pE4

There is a HindIII site approximately half-way along the fibre gene.Fusions with long fibres also contain this HindIII site. The fusion isintroduced at this site.

Recombination in vivo of plasmid pE4-ScFv with the adenovirus genome

To obtain virus particles expressing the ScFv on the penton fibresuitable cells, such as 293 cells, are cotransfected with plasmidpE4-ScFv and plasmid pFG173 as described in Mittal et al (1993) VinesRes. 28, 67-90, incorporated herein by reference. Since neither pFG173nor pE4-ScFv individually is able to generate virus progeny, ontransfection of 293 cells viable virus progeny are only produced by invivo recombination between these two plasmids resulting in rescue of thepenton fibre-ScFv fusion into the Ad5 genome.

293 cells are human transformed primary embryonal cells available fromthe ATCC under accession number ATCC CRL 1573.

The adenovirus particles made in this way express a NIP-binding ScFv ontheir surface. Such particles are useful in a two-step targetingapproach wherein a target-cell specific binding moiety, such as thoseidentified in Tables 1 and 2, are joined to NIP molecule and targeted toa cell. Once they have localized to the target cell within the patient,the adenovirus displaying NIP-binding ScFv is administered to thepatient and binds to the NIP.

EXAMPLE 3

Insertion of a cytotoxic gene into the E3 region of adenovirus Ad5

In preparation for rescue of the cytotoxic gene into the E3 region ofAd5, the cytotoxic coding sequences were first inserted into a cassettecontaining the SV40 early promoter and poly A addition sequences to giveplasmid pTOX as shown in FIG. 14.

To obtain virus with the cytotoxic gene and SV40 regulatory sequences inthe E3 region, 293 cells are cotransfected with plasmids pTOX and pFG173(FIG. 14). The plasmid pFG173 is constructed from pFG140, an infectiousplasmid containing the Ad5 d1309 genome in circular form by inserting akan^(r) gene at the EcoRI site as 75.9 m.u. as described in Grahm (1984)EMBO J. 3, 2917-2922 and Mitall et al (1993) Virus Res. 28, 67-90.

Since neither pFG173 nor pTOX individually is able to generateinfectious virus progeny, on transfection of 293 cells viable virusprogeny are only produced by in vivo recombination between these twoplasmids resulting in rescue of the E3 insert into the Ad5 genome.

Viral plaques obtained after cotransfection are isolated and expanded in293 cells and viral DNA was analyzed on an agarose gel after digestionwith HindIII. The structure of the desired Ad5-cytotoxic generecombinant is verified by the presence of diagnostic fragments. Onerecombinant is plaque purified and used for further study.

Legend to FIG. 14

The plasmid pFG173 contains the entire Ad5 genome, except for a 3.2 kbsequence spontaneously deleted between m.u. 75.9-84.9. Plasmids pTOX andpFG173 were used for cotransfection of 293 cells to rescue, by in vivorecombination, the cytotoxic gene flanked by SV40 regulatory sequencesin the E3 region of Ad5. The resulting Ad5-cytotoxic gene recombinantwas named Ad5-TOX. The relative positions of HindIII and XbaIrestriction sites of the Ad5-TOX genome are shown. The position andorientation of the SV40 promoter, the cytotoxic gene, and the SV40polyadenylation signal are shown below. Solid bars: luciferase gene;open bars: SV40 promoter and SV40 polyadenylation signal; hatched bars:amp^(r) and kan^(r) genes.

The cytotoxic gene is the cDNA for thymidine kinase.

Other cytotoxic genes are inserted into the E3 region of Ad5 in ananalogous manner.

EXAMPLE 4

Single chain Fv from the mouse monoclonal antibody HMFG1 and humanisedmonoclonal antibody Hu HMFG1

The nucleotide sequences encoding the V_(H) heavy chains and V_(K) lightchains of HMFG1 and Hu HMFG1 are shown in FIG. 15 and are given inVerhoeyen et al (1993) Immunology 78, 364-370, incorporated herein byreference.

Legend to FIG. 15

Nucleotide and amino acid sequences of mouse and reshaped HMFG1 variableregions. (a) Heavy chain variable region sequences for mouse andreshaped HMFG1 (Mo V_(H) -HMFG1 and Hu V_(H) -HMFG1); (b) mouse andreshaped light chain variable regions respectively (Mo V_(k) -HMFG1 andHu V_(k) -HMFG1). Amino acids numbering and definition of the CDR andframework regions are from Kabat et al (1987) Sequences of Proteins ofImmunological Interest, Edn 4, US Dept of Health and Human ServicesPublic Health Service, NIH, Bethesda, Md. 20892, USA.

The methods described by Bird et al (1988) Science 242, 423 or Huston etal (1988) Proc. Natl. Acad. Sci. USA 85, 5879 are applied to thenucleotide sequences described in FIG. 15 to generate genes encodingScFv for HMFG1 and ScFv for Hu HMFG1. These genes are fused individuallyinto the adenovirus penton fibre gene as described in Examples 1 and 2.

The amino acid sequences of the V_(H) and V_(L) chains of H17E2 aredisclosed in "Monoclonal antibodies--applications in clinical oncology",pages 37-43, 1991, A. A. Epenetos, ed., Chapman & Hall, UK.

Nucleotide sequences encoding the V_(H) and V_(L) chains are readilyderived from the amino acid sequence using the genetic code and an ScFvcan be made from the sequences using the methods of Bird et al or Hustonet al as described above.

    ______________________________________    Key to Sequence Listing                    SEQ ID No.                    Nucleotide                             Polypeptide    Name            Sequence Sequence    ______________________________________    Fusion A        1        2    Fusion B        3        4    Fusion C        5        6    Fusion D        7        8    Fusion E        9        10    Fusion F        11       12    Fusion G        13       14    Fusion H        15       16    Fusion I        17       18    Fusion J        19       20    Fusion K        21       22    Fusion L        23       24    Fusion M        25       26    Fusion N        27       28    Fusion O        29       30    Fusion P        31       32    Fusion Q        33       34    Fusion R        35       36    Fusion S        37       38    Fusion T        39       40    Fusion U        41       42    Fusion V        43       44    Xho-Eco         45       --    LEADHBACK       46       --    LEADbFOR        47       48    pRAS117         49       50    TAILbBACK       51       52    FIBRE3FOR       53       54    FIBRE6FOR       55       56    FIBRE9FOR       57       58    FIBRE12FOR      59       60    FIBRE15FOR      61       62    FIBRE18FOR      63       64    FIBRE21FOR      65       66    FIBRE22FOR      67       68    FIBREPFOR       69       70    pRAS111         71       72    MoV.sub.H       73       74    MoV.sub.k       75       76    HuV.sub.H       77       78    HuV.sub.k       79       80    ______________________________________

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 80    (2) INFORMATION FOR SEQ ID NO: 1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 30 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Adenovirus    (B) STRAIN: Ad5    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..30    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:    CCTCTAGTTACCTCCAATGTGCAGCTGCAG30    ProLeuValThrSerAsnValGlnLeuGln    1510    (2) INFORMATION FOR SEQ ID NO: 2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:    ProLeuValThrSerAsnValGlnLeuGln    1510    (2) INFORMATION FOR SEQ ID NO: 3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 30 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Adenovirus    (B) STRAIN: Ad5    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..30    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:    CTCTCTCTGGACGAGGCCGTGCAGCTGCAG30    LeuSerLeuAspGluAlaValGlnLeuGln    1510    (2) INFORMATION FOR SEQ ID NO: 4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:    LeuSerLeuAspGluAlaValGlnLeuGln    1510    (2) INFORMATION FOR SEQ ID NO: 5:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 30 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Adenovirus    (B) STRAIN: Ad5    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..30    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:    CCTCTCAAAAAAACCAAGGTGCAGCTGCAG30    ProLeuLysLysThrLysValGlnLeuGln    1510    (2) INFORMATION FOR SEQ ID NO: 6:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:    ProLeuLysLysThrLysValGlnLeuGln    1510    (2) INFORMATION FOR SEQ ID NO: 7:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 30 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Adenovirus    (B) STRAIN: Ad5    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..30    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:    CCCCTCACAGTTACCTCAGTGCAGCTGCAG30    ProLeuThrValThrSerValGlnLeuGln    1510    (2) INFORMATION FOR SEQ ID NO: 8:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:    ProLeuThrValThrSerValGlnLeuGln    1510    (2) INFORMATION FOR SEQ ID NO: 9:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 30 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Adenovirus    (B) STRAIN: Ad5    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..30    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:    CCTCTAATGGTCGCGGGCGTGCAGCTGCAG30    ProLeuMetValAlaGlyValGlnLeuGln    1510    (2) INFORMATION FOR SEQ ID NO: 10:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:    ProLeuMetValAlaGlyValGlnLeuGln    1510    (2) INFORMATION FOR SEQ ID NO: 11:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 30 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Adenovirus    (B) STRAIN: Ad5    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..30    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:    CCGCTAACCGTGCACGACGTGCAGCTGCAG30    ProLeuThrValHisAspValGlnLeuGln    1510    (2) INFORMATION FOR SEQ ID NO: 12:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:    ProLeuThrValHisAspValGlnLeuGln    1510    (2) INFORMATION FOR SEQ ID NO: 13:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 30 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Adenovirus    (B) STRAIN: Ad5    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..30    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:    CCCCTCACAGTGTCAGAAGTGCAGCTGCAG30    ProLeuThrValSerGluValGlnLeuGln    1510    (2) INFORMATION FOR SEQ ID NO: 14:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:    ProLeuThrValSerGluValGlnLeuGln    1510    (2) INFORMATION FOR SEQ ID NO: 15:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 30 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Adenovirus    (B) STRAIN: Ad5    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..30    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:    CTCACCACCACCGATAGCGTGCAGCTGCAG30    LeuThrThrThrAspSerValGlnLeuGln    1510    (2) INFORMATION FOR SEQ ID NO: 16:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:    LeuThrThrThrAspSerValGlnLeuGln    1510    (2) INFORMATION FOR SEQ ID NO: 17:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 30 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Adenovirus    (B) STRAIN: Ad5    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..30    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:    CCTCTAACTACTGCCACTGTGCAGCTGCAG30    ProLeuThrThrAlaThrValGlnLeuGln    1510    (2) INFORMATION FOR SEQ ID NO: 18:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:    ProLeuThrThrAlaThrValGlnLeuGln    1510    (2) INFORMATION FOR SEQ ID NO: 19:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 30 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Adenovirus    (B) STRAIN: Ad5    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..30    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:    CCCATTTATACACAAAATGTGCAGCTGCAG30    ProIleTyrThrGlnAsnValGlnLeuGln    1510    (2) INFORMATION FOR SEQ ID NO: 20:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:    ProIleTyrThrGlnAsnValGlnLeuGln    1510    (2) INFORMATION FOR SEQ ID NO: 21:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 30 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Adenovirus    (B) STRAIN: Ad5    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..30    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:    CATGTAACAGACGACCTAGTGCAGCTGCAG30    HisValThrAspAspLeuValGlnLeuGln    1510    (2) INFORMATION FOR SEQ ID NO: 22:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:    HisValThrAspAspLeuValGlnLeuGln    1510    (2) INFORMATION FOR SEQ ID NO: 23:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 30 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Adenovirus    (B) STRAIN: Ad5    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..30    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:    GGTGTGACTATTAATAATGTGCAGCTGCAG30    GlyValThrIleAsnAsnValGlnLeuGln    1510    (2) INFORMATION FOR SEQ ID NO: 24:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:    GlyValThrIleAsnAsnValGlnLeuGln    1510    (2) INFORMATION FOR SEQ ID NO: 25:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 30 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Adenovirus    (B) STRAIN: Ad5    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..30    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:    GGTTTTGATTCACAAGGCGTGCAGCTGCAG30    GlyPheAspSerGlnGlyValGlnLeuGln    1510    (2) INFORMATION FOR SEQ ID NO: 26:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:    GlyPheAspSerGlnGlyValGlnLeuGln    1510    (2) INFORMATION FOR SEQ ID NO: 27:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 30 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Adenovirus    (B) STRAIN: Ad5    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..30    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:    AGGATTGATTCTCAAAACGTGCAGCTGCAG30    ArgIleAspSerGlnAsnValGlnLeuGln    1510    (2) INFORMATION FOR SEQ ID NO: 28:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:    ArgIleAspSerGlnAsnValGlnLeuGln    1510    (2) INFORMATION FOR SEQ ID NO: 29:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 30 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Adenovirus    (B) STRAIN: Ad5    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..30    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:    TTTGATGCTCAAAACCAAGTGCAGCTGCAG30    PheAspAlaGlnAsnGlnValGlnLeuGln    1510    (2) INFORMATION FOR SEQ ID NO: 30:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:    PheAspAlaGlnAsnGlnValGlnLeuGln    1510    (2) INFORMATION FOR SEQ ID NO: 31:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 30 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Adenovirus    (B) STRAIN: Ad5    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..30    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:    CTTTTTATAAACTCAGCCGTGCAGCTGCAG30    LeuPheIleAsnSerAlaValGlnLeuGln    1510    (2) INFORMATION FOR SEQ ID NO: 32:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:    LeuPheIleAsnSerAlaValGlnLeuGln    1510    (2) INFORMATION FOR SEQ ID NO: 33:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 30 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Adenovirus    (B) STRAIN: Ad5    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..30    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:    TCAAACAATTCCAAAAACGTGCAGCTGCAG30    SerAsnAsnSerLysAsnValGlnLeuGln    1510    (2) INFORMATION FOR SEQ ID NO: 34:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:    SerAsnAsnSerLysAsnValGlnLeuGln    1510    (2) INFORMATION FOR SEQ ID NO: 35:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 30 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Adenovirus    (B) STRAIN: Ad5    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..30    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:    GGGTTGATGTTTGACGCTGTGCAGCTGCAG30    GlyLeuMetPheAspAlaValGlnLeuGln    1510    (2) INFORMATION FOR SEQ ID NO: 36:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36:    GlyLeuMetPheAspAlaValGlnLeuGln    1510    (2) INFORMATION FOR SEQ ID NO: 37:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 30 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Adenovirus    (B) STRAIN: Ad5    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..30    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:    CCTAATGCACCAAACACAGTGCAGCTGCAG30    ProAsnAlaProAsnThrValGlnLeuGln    1510    (2) INFORMATION FOR SEQ ID NO: 38:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38:    ProAsnAlaProAsnThrValGlnLeuGln    1510    (2) INFORMATION FOR SEQ ID NO: 39:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 30 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Adenovirus    (B) STRAIN: Ad5    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..30    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39:    CTAGAATTTGATTCAAACGTGCAGCTGCAG30    LeuGluPheAspSerAsnValGlnLeuGln    1510    (2) INFORMATION FOR SEQ ID NO: 40:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40:    LeuGluPheAspSerAsnValGlnLeuGln    1510    (2) INFORMATION FOR SEQ ID NO: 41:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 30 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Adenovirus    (B) STRAIN: Ad5    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..30    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41:    CTTAGTTTTGACAGCACAGTGCAGCTGCAG30    LeuSerPheAspSerThrValGlnLeuGln    1510    (2) INFORMATION FOR SEQ ID NO: 42:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42:    LeuSerPheAspSerThrValGlnLeuGln    1510    (2) INFORMATION FOR SEQ ID NO: 43:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 30 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Adenovirus    (B) STRAIN: Ad5    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..30    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43:    ATTGATAAGCTAACTTTGGTGCAGCTGCAG30    IleAspLysLeuThrLeuValGlnLeuGln    1510    (2) INFORMATION FOR SEQ ID NO: 44:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44:    IleAspLysLeuThrLeuValGlnLeuGln    1510    (2) INFORMATION FOR SEQ ID NO: 45:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Adenovirus    (B) STRAIN: Ad5    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45:    CTCGAGTAATAAGAATTC18    (2) INFORMATION FOR SEQ ID NO: 46:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 22 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46:    AGCTAAGCTTGCATGCAAATTC22    (2) INFORMATION FOR SEQ ID NO: 47:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 31 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..27    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47:    CCAGCGATGGCCAGATCTCAGCTGCAGAGCT31    ProAlaMetAlaArgSerGlnLeuGln    15    (2) INFORMATION FOR SEQ ID NO: 48:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 9 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48:    ProAlaMetAlaArgSerGlnLeuGln    15    (2) INFORMATION FOR SEQ ID NO: 49:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 132 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 40..132    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49:    AAGCTTGCATGCAAATTCTATTTCAAGGAGACAGTCATAATGAAATACCTATTG54    MetLysTyrLeuLeu    15    CCTACGGCAGCCGCTGGATTGTTATTACTCGCTGCCCAACCAGCGATG102    ProThrAlaAlaAlaGlyLeuLeuLeuLeuAlaAlaGlnProAlaMet    101520    GCCAGATCTCAGCTGCAGGTCGACGGATCC132    AlaArgSerGlnLeuGlnValAspGlySer    2530    (2) INFORMATION FOR SEQ ID NO: 50:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 31 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50:    MetLysTyrLeuLeuProThrAlaAlaAlaGlyLeuLeuLeuLeuAla    151015    AlaGlnProAlaMetAlaArgSerGlnLeuGlnValAspGlySer    202530    (2) INFORMATION FOR SEQ ID NO: 51:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 28 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 5..28    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 51:    AGCTAGATCTATGAAGCGCGCAAGACCG28    ArgSerMetLysArgAlaArgPro    15    (2) INFORMATION FOR SEQ ID NO: 52:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 8 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 52:    ArgSerMetLysArgAlaArgPro    15    (2) INFORMATION FOR SEQ ID NO: 53:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 41 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..33    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 53:    CCTCTCAAAAAAACCAAGCAGGTGCAGCTGCAGCAGCCTGG41    ProLeuLysLysThrLysGlnValGlnLeuGln    1510    (2) INFORMATION FOR SEQ ID NO: 54:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 11 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 54:    ProLeuLysLysThrLysGlnValGlnLeuGln    1510    (2) INFORMATION FOR SEQ ID NO: 55:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 42 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 2..34    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 55:    CCCGCTAACCGTGCACGACCAGGTGCAGCTGCAGCAGCCTGG42    ProLeuThrValHisAspGlnValGlnLeuGln    1510    (2) INFORMATION FOR SEQ ID NO: 56:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 11 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 56:    ProLeuThrValHisAspGlnValGlnLeuGln    1510    (2) INFORMATION FOR SEQ ID NO: 57:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 41 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..33    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 57:    CCTCTAACTACTGCCACTCAGGTGCAGCTGCAGCAGCCTGG41    ProLeuThrThrAlaThrGlnValGlnLeuGln    1510    (2) INFORMATION FOR SEQ ID NO: 58:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 11 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 58:    ProLeuThrThrAlaThrGlnValGlnLeuGln    1510    (2) INFORMATION FOR SEQ ID NO: 59:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 41 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..33    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 59:    GGTGTGACTATTAATAATCAGGTGCAGCTGCAGGACCCTGG41    GlyValThrIleAsnAsnGlnValGlnLeuGln    1510    (2) INFORMATION FOR SEQ ID NO: 60:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 11 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 60:    GlyValThrIleAsnAsnGlnValGlnLeuGln    1510    (2) INFORMATION FOR SEQ ID NO: 61:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 41 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..36    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 61:    CCGTTTGATGCTCAAAACCAACAGGTGCAGCTGCAGCAGCC41    ProPheAspAlaGlnAsnGlnGlnValGlnLeuGln    1510    (2) INFORMATION FOR SEQ ID NO: 62:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 12 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 62:    ProPheAspAlaGlnAsnGlnGlnValGlnLeuGln    1510    (2) INFORMATION FOR SEQ ID NO: 63:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 38 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..33    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 63:    GGGTTGATGTTTGACGCTCAGGTGCAGCTGCAGCAGCC38    GlyLeuMetPheAspAlaGlnValGlnLeuGln    1510    (2) INFORMATION FOR SEQ ID NO: 64:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 11 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 64:    GlyLeuMetPheAspAlaGlnValGlnLeuGln    1510    (2) INFORMATION FOR SEQ ID NO: 65:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 40 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 3..35    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 65:    GCCTTAGTTTTGACAGCACACAGGTGCAGCTGCAGCAGCC40    LeuSerPheAspSerThrGlnValGlnLeuGln    1510    (2) INFORMATION FOR SEQ ID NO: 66:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 11 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 66:    LeuSerPheAspSerThrGlnValGlnLeuGln    1510    (2) INFORMATION FOR SEQ ID NO: 67:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 50 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..45    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 67:    GGAAACAAAAATAATGATAAGCTAACTTTGCAGGTGCAGCTGCAG45    GlyAsnLysAsnAsnAspLysLeuThrLeuGlnValGlnLeuGln    151015    CAGCC50    (2) INFORMATION FOR SEQ ID NO: 68:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 15 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 68:    GlyAsnLysAsnAsnAspLysLeuThrLeuGlnValGlnLeuGln    151015    (2) INFORMATION FOR SEQ ID NO: 69:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 43 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 3..17    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 69:    CATACATTGCCCAAGAATAACAGGTGCAGCTGCAGCAGCCTGG43    TyrIleAlaGlnGlu    15    (2) INFORMATION FOR SEQ ID NO: 70:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 5 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 70:    TyrIleAlaGlnGlu    15    (2) INFORMATION FOR SEQ ID NO: 71:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 858 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 40..846    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 71:    AAGCTTGCATGCAAATTCTATTTCAAGGAGACAGTCATAATGAAATACCTATTG54    MetLysTyrLeuLeu    15    CCTACGGCAGCCGCTGGATTGTTATTACTCGCTGCCCAACCAGCGATG102    ProThrAlaAlaAlaGlyLeuLeuLeuLeuAlaAlaGlnProAlaMet    101520    GCCCAGGTGCAGCTGCAGCAGCCTGGGGCTGAGCTTGTGAAGCCTGGG150    AlaGlnValGlnLeuGlnGlnProGlyAlaGluLeuValLysProGly    253035    GCTTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGC198    AlaSerValLysLeuSerCysLysAlaSerGlyTyrThrPheThrSer    404550    TACTGGATGCACTGGGTGAAGCAGAGGCCTGGACGAGGCCTTGAGTGG246    TyrTrpMetHisTrpValLysGlnArgProGlyArgGlyLeuGluTrp    556065    ATTGGAAGGATTGATCCTAATAGTGGTGGTACTAAGTACAATGAGAAG294    IleGlyArgIleAspProAsnSerGlyGlyThrLysTyrAsnGluLys    70758085    TTCAAGAGCAAGGCCACACTGACTGTAGACAAACCCTCCAGCACAGCC342    PheLysSerLysAlaThrLeuThrValAspLysProSerSerThrAla    9095100    TACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTAT390    TyrMetGlnLeuSerSerLeuThrSerGluAspSerAlaValTyrTyr    105110115    TGTGCAAGATACGATTACTACGGTAGTAGCTACTTTGACTACTGGGGC438    CysAlaArgTyrAspTyrTyrGlySerSerTyrPheAspTyrTrpGly    120125130    CAAGGGACCACGGTCACCGTCTCCTCAGGTGGAGGCGGTTCAGGCGGA486    GlnGlyThrThrValThrValSerSerGlyGlyGlyGlySerGlyGly    135140145    GGTGGCTCTGGCGGTGGCGGATCCCAGGCTGTTGTGACTCAGGAATCT534    GlyGlySerGlyGlyGlyGlySerGlnAlaValValThrGlnGluSer    150155160165    GCACTCACCACATCACCTGGTGAAACAGTCACACTCACTTGTCGCTCA582    AlaLeuThrThrSerProGlyGluThrValThrLeuThrCysArgSer    170175180    AGTACTGGGGCTGTTACAACTAGTAACTATGCCAACTGGGTCCAAGAA630    SerThrGlyAlaValThrThrSerAsnTyrAlaAsnTrpValGlnGlu    185190195    AAACCAGATCATTTATTCACTGGTCTAATAGGTGGTACCAACAACCGA678    LysProAspHisLeuPheThrGlyLeuIleGlyGlyThrAsnAsnArg    200205210    GCTCCAGGTGTTCCTGCCAGATTCTCAGGCTCCCTGATTGGAGACAAG726    AlaProGlyValProAlaArgPheSerGlySerLeuIleGlyAspLys    215220225    GCTGCCCTCACCATCACAGGGGCACAGACTGAGGATGAGGCAATATAT774    AlaAlaLeuThrIleThrGlyAlaGlnThrGluAspGluAlaIleTyr    230235240245    TTCTGTGCTCTATGGTACAGCAACCACTGGGTGTTCGGTGGAGGAACC822    PheCysAlaLeuTrpTyrSerAsnHisTrpValPheGlyGlyGlyThr    250255260    AAACTGACTGTCCTAGGTCTCGAGTAATAAGAATTC858    LysLeuThrValLeuGlyLeuGlu    265    (2) INFORMATION FOR SEQ ID NO: 72:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 269 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 72:    MetLysTyrLeuLeuProThrAlaAlaAlaGlyLeuLeuLeuLeuAla    151015    AlaGlnProAlaMetAlaGlnValGlnLeuGlnGlnProGlyAlaGlu    202530    LeuValLysProGlyAlaSerValLysLeuSerCysLysAlaSerGly    354045    TyrThrPheThrSerTyrTrpMetHisTrpValLysGlnArgProGly    505560    ArgGlyLeuGluTrpIleGlyArgIleAspProAsnSerGlyGlyThr    65707580    LysTyrAsnGluLysPheLysSerLysAlaThrLeuThrValAspLys    859095    ProSerSerThrAlaTyrMetGlnLeuSerSerLeuThrSerGluAsp    100105110    SerAlaValTyrTyrCysAlaArgTyrAspTyrTyrGlySerSerTyr    115120125    PheAspTyrTrpGlyGlnGlyThrThrValThrValSerSerGlyGly    130135140    GlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySerGlnAlaVal    145150155160    ValThrGlnGluSerAlaLeuThrThrSerProGlyGluThrValThr    165170175    LeuThrCysArgSerSerThrGlyAlaValThrThrSerAsnTyrAla    180185190    AsnTrpValGlnGluLysProAspHisLeuPheThrGlyLeuIleGly    195200205    GlyThrAsnAsnArgAlaProGlyValProAlaArgPheSerGlySer    210215220    LeuIleGlyAspLysAlaAlaLeuThrIleThrGlyAlaGlnThrGlu    225230235240    AspGluAlaIleTyrPheCysAlaLeuTrpTyrSerAsnHisTrpVal    245250255    PheGlyGlyGlyThrLysLeuThrValLeuGlyLeuGlu    260265    (2) INFORMATION FOR SEQ ID NO: 73:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 354 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Mouse    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..354    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 73:    CAGGTTCAGCTGCAGCAGTCTGGAGCTGAGCTGATGAAGCCTGGGGCC48    GlnValGlnLeuGlnGlnSerGlyAlaGluLeuMetLysProGlyAla    151015    TCAGTGAAGATATCCTGCAAGGCTACTGGCTACACATTCAGTGCCTAC96    SerValLysIleSerCysLysAlaThrGlyTyrThrPheSerAlaTyr    202530    TGGATAGAGTGGGTAAAGCAGAGGCCTGGACATGGCCTTGAGTGGATT144    TrpIleGluTrpValLysGlnArgProGlyHisGlyLeuGluTrpIle    354045    GGAGAGATTTTACCTGGAAGTAATAATTCTAGATACAATGAGAAGTTC192    GlyGluIleLeuProGlySerAsnAsnSerArgTyrAsnGluLysPhe    505560    AAGGGCAAGGCCACATTCACTGCTGATACATCCTCCAACACAGCCTAC240    LysGlyLysAlaThrPheThrAlaAspThrSerSerAsnThrAlaTyr    65707580    ATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCCGTCTATTACTGT288    MetGlnLeuSerSerLeuThrSerGluAspSerAlaValTyrTyrCys    859095    TCAAGGTCCTACGACTTTGCCTGGTTTGCTTACTGGGGCCAAGGGACT336    SerArgSerTyrAspPheAlaTrpPheAlaTyrTrpGlyGlnGlyThr    100105110    CCGGTCACTGTCTCTGCA354    ProValThrValSerAla    115    (2) INFORMATION FOR SEQ ID NO: 74:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 118 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 74:    GlnValGlnLeuGlnGlnSerGlyAlaGluLeuMetLysProGlyAla    151015    SerValLysIleSerCysLysAlaThrGlyTyrThrPheSerAlaTyr    202530    TrpIleGluTrpValLysGlnArgProGlyHisGlyLeuGluTrpIle    354045    GlyGluIleLeuProGlySerAsnAsnSerArgTyrAsnGluLysPhe    505560    LysGlyLysAlaThrPheThrAlaAspThrSerSerAsnThrAlaTyr    65707580    MetGlnLeuSerSerLeuThrSerGluAspSerAlaValTyrTyrCys    859095    SerArgSerTyrAspPheAlaTrpPheAlaTyrTrpGlyGlnGlyThr    100105110    ProValThrValSerAla    115    (2) INFORMATION FOR SEQ ID NO: 75:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 342 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Mouse    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..342    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 75:    GACATTGTGATGTCACAGTCTCCATCCTCCCTAGCTGTGTCAGTTGGA48    AspIleValMetSerGlnSerProSerSerLeuAlaValSerValGly    151015    GAGAAGGTTACTATGAGCTGCAAGTCCAGTCAGAGCCTTTTATATAGT96    GluLysValThrMetSerCysLysSerSerGlnSerLeuLeuTyrSer    202530    AGCAATCAAAAGATCTACTTGGCCTGGTACCAGCAGAAACCAGGGCAG144    SerAsnGlnLysIleTyrLeuAlaTrpTyrGlnGlnLysProGlyGln    354045    TCTCCTAAACTGCTGATTTACTGGGCATCCACTAGGGAATCTGGGGTC192    SerProLysLeuLeuIleTyrTrpAlaSerThrArgGluSerGlyVal    505560    CCTGATCGCTTCACAGGCGGTGGATCTGGGACAGATTTCACTCTCACC240    ProAspArgPheThrGlyGlyGlySerGlyThrAspPheThrLeuThr    65707580    ATCAGCAGTGTGAAGGCTGAAGACCTGGCAGTTTATTACTGTCAGCAA288    IleSerSerValLysAlaGluAspLeuAlaValTyrTyrCysGlnGln    859095    TATTATAGATATCCTCGGACGTTCGGTGGAGGCACCAAGCTGGAAATC336    TyrTyrArgTyrProArgThrPheGlyGlyGlyThrLysLeuGluIle    100105110    AAACGG342    LysArg    (2) INFORMATION FOR SEQ ID NO: 76:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 114 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 76:    AspIleValMetSerGlnSerProSerSerLeuAlaValSerValGly    151015    GluLysValThrMetSerCysLysSerSerGlnSerLeuLeuTyrSer    202530    SerAsnGlnLysIleTyrLeuAlaTrpTyrGlnGlnLysProGlyGln    354045    SerProLysLeuLeuIleTyrTrpAlaSerThrArgGluSerGlyVal    505560    ProAspArgPheThrGlyGlyGlySerGlyThrAspPheThrLeuThr    65707580    IleSerSerValLysAlaGluAspLeuAlaValTyrTyrCysGlnGln    859095    TyrTyrArgTyrProArgThrPheGlyGlyGlyThrLysLeuGluIle    100105110    LysArg    (2) INFORMATION FOR SEQ ID NO: 77:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 354 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..354    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 77:    CAGGTGCAGCTGGTGCAGTCTGGGGCAGAGGTGAAAAAGCCTGGGGCC48    GlnValGlnLeuValGlnSerGlyAlaGluValLysLysProGlyAla    151015    TCAGTGAAGGTGTCCTGCAAGGCTTCTGGCTACACCTTCAGTGCCTAC96    SerValLysValSerCysLysAlaSerGlyTyrThrPheSerAlaTyr    202530    TGGATAGAGTGGGTGCGCCAGGCTCCAGGAAAGGGCCTCGAGTGGGTC144    TrpIleGluTrpValArgGlnAlaProGlyLysGlyLeuGluTrpVal    354045    GGAGAGATTTTACCTGGAAGTAATAATTCTAGATACAATGAGAAGTTC192    GlyGluIleLeuProGlySerAsnAsnSerArgTyrAsnGluLysPhe    505560    AAGGGCCGAGTGACAGTCACTAGAGACACATCCACAAACACAGCCTAC240    LysGlyArgValThrValThrArgAspThrSerThrAsnThrAlaTyr    65707580    ATGGAGCTCAGCAGCCTGAGGTCTGAGGACACAGCCGTCTATTACTGT288    MetGluLeuSerSerLeuArgSerGluAspThrAlaValTyrTyrCys    859095    GCAAGATCCTACGACTTTGCCTGGTTTGCTTACTGGGGCCAAGGGACT336    AlaArgSerTyrAspPheAlaTrpPheAlaTyrTrpGlyGlnGlyThr    100105110    CTGGTCACAGTCTCCTCA354    LeuValThrValSerSer    115    (2) INFORMATION FOR SEQ ID NO: 78:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 118 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 78:    GlnValGlnLeuValGlnSerGlyAlaGluValLysLysProGlyAla    151015    SerValLysValSerCysLysAlaSerGlyTyrThrPheSerAlaTyr    202530    TrpIleGluTrpValArgGlnAlaProGlyLysGlyLeuGluTrpVal    354045    GlyGluIleLeuProGlySerAsnAsnSerArgTyrAsnGluLysPhe    505560    LysGlyArgValThrValThrArgAspThrSerThrAsnThrAlaTyr    65707580    MetGluLeuSerSerLeuArgSerGluAspThrAlaValTyrTyrCys    859095    AlaArgSerTyrAspPheAlaTrpPheAlaTyrTrpGlyGlnGlyThr    100105110    LeuValThrValSerSer    115    (2) INFORMATION FOR SEQ ID NO: 79:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 342 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..342    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 79:    GACATCCAGATGACCCAGAGCCCAAGCAGCCTGAGCGCCAGCGTGGGT48    AspIleGlnMetThrGlnSerProSerSerLeuSerAlaSerValGly    151015    GACAGAGTGACCATCACCTGTAAGTCCAGTCAGAGCCTTTTATATAGT96    AspArgValThrIleThrCysLysSerSerGlnSerLeuLeuTyrSer    202530    AGCAATCAAAAGATCTACTTGGCCTGGTACCAGCAGAAGCCAGGTAAG144    SerAsnGlnLysIleTyrLeuAlaTrpTyrGlnGlnLysProGlyLys    354045    GCTCCAAAGCTGCTGATCTACTGGGCATCCACTAGGGAATCTGGTGTG192    AlaProLysLeuLeuIleTyrTrpAlaSerThrArgGluSerGlyVal    505560    CCAAGCAGATTCAGCGGTAGCGGTAGCGGTACCGACTTCACCTTCACC240    ProSerArgPheSerGlySerGlySerGlyThrAspPheThrPheThr    65707580    ATCAGCAGCCTCCAGCCAGAGGACATCGCCACCTACTACTGCCAGCAA288    IleSerSerLeuGlnProGluAspIleAlaThrTyrTyrCysGlnGln    859095    TATTATAGATATCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATC336    TyrTyrArgTyrProArgThrPheGlyGlnGlyThrLysValGluIle    100105110    AAACGT342    LysArg    (2) INFORMATION FOR SEQ ID NO: 80:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 114 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 80:    AspIleGlnMetThrGlnSerProSerSerLeuSerAlaSerValGly    151015    AspArgValThrIleThrCysLysSerSerGlnSerLeuLeuTyrSer    202530    SerAsnGlnLysIleTyrLeuAlaTrpTyrGlnGlnLysProGlyLys    354045    AlaProLysLeuLeuIleTyrTrpAlaSerThrArgGluSerGlyVal    505560    ProSerArgPheSerGlySerGlySerGlyThrAspPheThrPheThr    65707580    IleSerSerLeuGlnProGluAspIleAlaThrTyrTyrCysGlnGln    859095    TyrTyrArgTyrProArgThrPheGlyGlnGlyThrLysValGluIle    100105110    LysArg    __________________________________________________________________________

We claim:
 1. An adenovirus, or adenovirus-like particle, haivng a pentonfibre comprising a modified binding specificity conferred by a bindingmoiety which is heterologous to the adenovirus and is incorporated as afusion protein with the fibre protein allowing the adenovirus oradenovirus-like particle to bind to a target cell which is not thenatural host cell of the virus, characterized in that the said pentonfibre is modified by the insertion or deletion or substitution of aminoacid residues that disrupt the host-cell binding function so that theadenovirus or adenovirus-like particle is substantially incapable ofbinding the natural host cell.
 2. An adenovirus or adenovirus-likeparticle according to claim 1 wherein the binding moiety is a monoclonalantibody, an ScFv, a dAb, or a minimal recognition unit of an antibody.3. An adenovirus or adenovirus-like or virus-like particle according toclaim 1 wherein the binding moiety is at least part of a ligand of atarget cell-specific cell-surface receptor.
 4. An adenovirus oradenovirus-like particle according, to claim 2 wherein the bindingmoiety recognizes a target cell specific surface antigen.
 5. Anadenovirus or adenovirus-like particle according to claim 3 wherein thetarget cell-specific cell-surface receptor is any one of GnRH receptor,MSH receptor and somatostatin receptor.
 6. An adenovirus, oradenovirus-like particle, containing nucleic acid, according to claim 1wherein the said adenovirus or adenovirus-like particle is adapted todeliver the said nucleic acid to the target cell.
 7. An adenovirus oradenovirus-like particle according to claim 1 wherein the binding moietyis fused to the penton fibre protein at any one or more of the junctionsof the repetitive units of the shaft.
 8. An adenovirus oradenovirus-like particle according to claim 7 wherein the binding moietyis a ScFv.
 9. An adenovirus or adenovirus-like particle according toclaim 8 wherein the ScFv binds to a tumor cell antigen.
 10. A nucleotidesequence encoding the penton fibre modified as defined in claim
 1. 11. Anucleotide sequence defined in claim 10 additionally, comprising theremainder of the genome of the adenovirus or adenovirus-like particle.12. A nucleotide sequence encoding an adenovirus or adenovirus-likeparticle according to claim
 1. 13. A method for producing an adenovirusor adenovirus-like particle of claim 1 in cell culture, the methodcomprising ( 1) genetically modifying an adenovirus or adenovirus-likeparticle to produce a binding moiety which is incorporated as a fusionprotein with the fibre protein, (2) infecting cells with the geneticallymodified adenovirus or adenovirus-like particle, (3) culturing the cellsuntil the adenovirus or adenovirus-like particle reaches a sufficientlyhigh titre and (4) harvesting and substantially purifying thegenetically modified adenovirus or adenovirus-like particle.
 14. Anadenovirus or adenovirus-like particle having a penton fibre, comprisinga modified binding specificity conferred by a binding moiety allowingthe adenovirus or adenovirus-like particle to bind to a target cell,characterized in that the penton fibre protein is substantiallyincapable of binding the natural host cell and the binding moiety isfused to the penton fibre protein at any one or more of the junctions ofthe repetition units of the shaft.